The enzyme-linked immunosorbent assay (ELISA), is a technique commonly used to detect disease markers in body fluids of humans and animals (J. R. Crowther (1995) “Elisa: Theory and Practice”, Totowa, N.J.; Humana Press). Such disease markers, generally referred to as antigens, are identified via capture on a surface with the aid of a surface-bound antibody that is highly selective toward molecular recognition and formation of a chemical complex with the said antigen, followed by a quantitative chemical analysis sequence.
In an ELISA, antigen that is captured by a specific antibody in the form of a surface-bound complex may be detected and quantified by means of an analytical chemistry protocol (see, e.g., FIG. 1). For example, a micro-well plate array may be used (101) with a primary antibody attached to the surface of the plate (102) that selectively recognizes and attaches to the antigen (103). The signal amplification sequence may start by selectively attaching a secondary antibody (104) (which is said to be “enzyme-linked” i.e., has an enzyme (e.g., horseradish peroxidase) attached to it, 105) and that recognizes the exposed portions of the antigen molecule that are left free after antigen attachment to the surface-bound primary antibody. The enzyme may be a catalyst capable of converting substrates into detectable products (e.g., colored products). Thus, a solution containing an adequate substrate may be added to the micro-wells having the surface-bound polymolecular complex consisting of species 102 through 104. After an incubation period, the amount of captured antigen may be determined by quantifying the amount of converted substrate.
Despite the fact that enzymes, for example horseradish peroxidase, are capable of converting roughly 1,000,000 substrate molecules into their color signal-emitting products per second, there are at least two limitations in the current art. First, the synthesis of the secondary antibody-enzyme complex may be laborious, costly or simply be impractical or impossible to carry out in certain cases. Second, a secondary antibody-enzyme complex only carries one signal-amplifying molecule (e.g., an enzyme) per single surface-captured antigen molecule. Furthermore, chemical interferences can affect the biochemical reaction. As such, the ratio of signal-amplifying molecule to antigen (e.g., the stoichiometric signal-amplifying molecule:antigen ratio) is one and results in an intrinsic biochemical limit for signal amplification. It is thus desirable to devise an ELISA antigen detection system that does not rely on the biochemical synthesis of a secondary antibody-enzyme complex.